DNA polymerase I (Pol I) of E. coli is an enzyme that plays an important role in the cell both during replication, in the processing of Okazaki fragments, and in the repair of damaged DNA. It is arguably the best model system for the study of the molecular details of polymerase action, and for an investigation of the way in which processive enzymes can co-ordinate synthesis with movement along a macromolecule. Our recent work has provided the tools for a detailed analysis of the mechanism of action of Pol I: we have determined the sequence of the structural gene (po1A), constructed strains which overproduce a functional polymerase fragment suitable for structural studies, and established a system for the rapid mapping, cloning and sequencing of po1A mutants. Most of the work described in this proposal is designed to complement the X-ray crystallographic studies of our collaborators, by locating the DNA binding site and assigning functions to regions of the Pol I molecule. We shall define the interaction between Pol I and its DNA substrate both nby affinity labeling the protein, and by chemical and enzymatic probing of contacts on the DNA molecule. We shall identify functionally important regions of Pol I by sequencing and enzymatic studies of po1A mutants. In addition, we intend to examine aspects of the biological role of the po1A gene, determining whether the gene is essential, what factors control the intracellular level of Pol I, and whether Pol I functions in association with other proteins. While this project has no direct health-related application, it is clear that an improved understanding of those processes in DNA replication and repair that maintain the integrity of the genome should eventually provide insights into the mechanisms of mutagenesis and thus of carcinogenesis.